Marc R. Freeman, Ph.D. - US grants

DESCRIPTION (Adapted from applicant's abstract): Dare mutants behave abnormally in response to olfactory repellents and have defects in male courtship behavior. The long-term goal of this project is to characterize the role of dare in the function and development of the Drosophila olfactory system. Dare encodes the Drosophila homologue of adrenodoxin reductase (AR), which has been shown in mammals to be essential for the key, rate limiting step in the synthesis of all steroid hormones. Dare is the first mutant with a lesion in the steroid biosynthetic pathway and links steroid hormones with sensory behavior. Several new dare alleles will be tested in a variety of behavioral and electrophysiological olfactory paradigms. Nervous system morphology in dare mutant animals will be observed at several developmental stages in an attempt to identify the nature, timing, and extent of any abnormalities. I will attempt to rescue behavioral and developmental defects using a transformant line harboring a heat shock-inducible AR cDNA construct (hs-AR). In the absence of full phenotypic rescue by hs-AR, I will generate flies harboring a genomic fragment which covers the AR gene for similar experiments and begin a screen for new dare alleles. After dare has been rigorously identified as AR, developmental Northern analysis and in situ hybridizations to RNA in tissue sections will be used to determine the pattern and developmental time course of dare expression. Time permitting, I will screen for a temperature-sensitive allele of dare and attempt to rescue male courtship defects by the directed expression of dare in male pheromone-producing tissues.

[unreadable] DESCRIPTION (provided by applicant): Glia are the primary immune cell type in the nervous system. In response to neural infection or trauma they becoming "reactive", undergoing stereotypical changes in gene expression and morphology. However, the molecular details of glial responses to neural injury or death are poorly understood. We study glial immune functions in Drosophila because it has well-defined glial subtypes that resemble mammalian glia, and it is amenable to genetic analysis. This proposal focuses on the role of Drosophila glia and the newly-identified Drpr receptor in glial engulfment of dead neurons, and glial responses to neural injury in the CNS. Drpr encodes an engulfment receptor essential for glial removal of neuronal cell corpses; the Drpr receptor is also potently transcriptionally upregulated in glia after the axons they ensheath are severed. We will take a molecular genetic approach to understand how Drpr functions in glial responses to neural injury, glial engulfment of injured axons, and additional mechanisms of neuron-glia interactions after neural trauma. Our specific aims are: 1) Characterize Draper functions in the embryonic CNS: We will define the role for Drpr and specific Drpr receptor isoforms in cell corpse removal and glial morphogenesis in the Drosophila embryonic CNS. 2) Define glial responses to neural injury and roles for Draper in removing injured axons: We will define morphological and molecular changes exhibited by glia in response to neural injury, and determine the requirements for Drpr and glia in the removal of injured axons. 3) Define the cellular and molecular action of Wlds protein: The Wallerian degeneration slow (Wlds) protein protects injured axons from degeneration by unknown mechanisms. We found that Wlds can also spare severed Drosophila axons from degeneration. We will explore Wlds-mediated protection of Drosophila axons, and the consequences of Wlds expression on glial responses to neural injury. Mechanisms of neuron-glia communication after neural injury are likely well-conserved in flies and mice, since Drosophila glia also become reactive and Wlds can protect severed axons in flies. Characterizing genes that regulate neuronal and glial function in response to CNS trauma is essential to identify new avenues for the treatment of CNS injury and neurological disease. [unreadable] [unreadable]

Glia are the primary immune cell type in the nervous system. In response to neural infection or trauma they becoming "reactive", undergoing stereotypical changes in gene expression and morphology. However, the molecular details of glial responses to neural injury or death are poorly understood. We study glial immune functions in Drosophila because it has well-defined glial subtypes that resemble mammalian glia, and it is amenable to genetic analysis. This proposal focuses on the role of Drosophila glia and the newly-identified Drpr receptor in glial engulfment of dead neurons, and glial responses to neural injury in the CNS. Drpr encodes an engulfment receptor essential for glial removal of neuronal cell corpses; the Drpr receptor is also potently transcriptionally upregulated in glia after the axons they ensheath are severed. We will take a molecular genetic approach to understand how Drpr functions in glial responses to neural injury, glial engulfment of injured axons, and additional mechanisms of neuron-glia interactions after neural trauma. Our specific aims are: 1) Characterize Draper functions in the embryonic CNS: We will define the role for Drpr and specific Drpr receptor isoforms in cell corpse removal and glial morphogenesis in the Drosophila embryonic CNS. 2) Define glial responses to neural injury and roles for Draper in removing injured axons: We will define morphological and molecular changes exhibited by glia in response to neural injury, and determine the requirements for Drpr and glia in the removal of injured axons. 3) Define the cellular and molecular action of Wlds protein: The Wallerian degeneration slow (Wlds) protein protects injured axons from degeneration by unknown mechanisms. We found that Wlds can also spare severed Drosophila axons from degeneration. We will explore Wlds-mediated protection of Drosophila axons, and the consequences of Wlds expression on glial responses to neural injury. Mechanisms of neuron-glia communication after neural injury are likely well-conserved in flies and mice, since Drosophila glia also become reactive and Wlds can protect severed axons in flies. Characterizing genes that regulate neuronal and glial function in response to CNS trauma is essential to identify new avenues for the treatment of CNS injury and neurological disease.

DESCRIPTION (provided by applicant): Axon degeneration occurs after nervous system injury and during neurodegenerative diseases but very little is known about how injured or diseased axons destroy themselves. Recent work on the mouse Wallerian degeneration slow molecule (Wlds), which potently protects severed axons from degeneration, has revealed that axon degeneration is an active process of axon auto-destruction. Amazingly, Wlds can also suppress axon degeneration after chemical insult and delay disease onset in a number of mouse models of human neurodegenerative disease. Wlds is therefore a broadly neuroprotective molecule and understanding its molecular action is of paramount importance. We have developed the first Drosophila model to study injury-induced axon degeneration and shown that mouse Wlds can also potently suppress axon degeneration in severed Drosophila axons. These data indicate that the molecular mechanism that drive axon auto-destruction after injury are well-conserved in Drosophila and mammals, and open the door to powerful molecular-genetic approaches only available in Drosophila to study axon auto-destruction. In this proposal we will: (1) define the domains of the Wlds protein essential for it to protect axons; (2) determine whether Wlds interacts with the ubiquitin proteasome, NAD biosynthetic, or apoptotic machinery to block axon auto-destruction; and (3) perform the first ever forward genetic screens for mutation that block axon degeneration after injury or Wlds neuroprotective function. These studies represent the beginning of a long-term comprehensive effort to understand how axons destroy themselves after injury, and how Wlds impinges upon these pathways. We expect our findings to have a major impact on our understanding of axon degeneration after injury or during disease in humans, and the novel molecules we identify will be excellent candidates for therapeutic intervention in human axonopathies. PUBLIC HEALTH RELEVANCE: After brain injury or during neurological disease neuronal fibers degenerate, connections in the brain are lost, and neural function is irreversibly compromised. We are studying the cellular action of an extraordinary molecule, WldS, which suppresses this loss of neuronal fibers. Our work will identify many new molecules that will be targets for treatment of patients after brain injury or during neurological disease.

DESCRIPTION (provided by applicant): Glial cells are major constituents of the nervous system, and an array of devastating diseases, such as childhood periventricular leukomalacia, Alexanders Disease, and demyelinating diseases such as multiple sclerosis, arise from glial malfunction. Although glial cells have been recently recognized as playing key roles in neuronal function, sculpting synaptic connections, and providing essential trophic factors to neurons, there are major gaps in our understanding of the molecular mechanisms mediating their diverse actions. This proposal employs a highly tractable system, the glutamatergic Drosophila neuromuscular junction (NMJ), to investigate major questions in glial cell biology with exquisite cellular detail. Our preliminary data provides compelling evidence for a role of glial cells in the plasticity of NMJs, regulating an important pathway required for the differentiation of synapses, and sculpting synaptic connections by a process of pruning. In this project we will (Aim 1) test the hypothesis that glial cells play a primary role in the extension and retraction of synaptic boutons during NMJ expansion, (Aim 2) test the hypothesis that glial cells regulate a Wnt pathway at the NMJ and that this regulation is essential for NMJ development, and (Aim 3) determine the role of the Draper signaling pathway in sculpting synaptic connections at the NMJ. We expect that these studies will provide fundamental insights into the cellular interactions between synapses and glia in live animals and unravel molecular mechanisms by which glia actively sculpt synaptic connections. Project Narrative Glial cells, the major cell type in the human brain, have emerged as important regulators of brain development and physiology, and a number of devastating neurological diseases, such as multiple sclerosis or glioma, are associated with glial dysfunction. This proposal will explore how glia (in live animals) modulate the formation and modification of synapses, the basic functional units through which neurons communicate with other cell types. Our work will provide fundamental knowledge regarding how neurons and glia communicate during the modification of synapses, and is expected to provide important insights into how glial dysfunction might cause disease.

DESCRIPTION (provided by applicant): Astrocytes are the major cell type in the human brain and in recent years have emerged as critical regulators of neural circuit development, function, plasticity, and maintenance. An array of devastating neurological conditions result from astrocyte dysfunction including childhood periventricular leukomalacia, amyotrophic lateral sclerosis, and gliomas, one of the most deadly forms of cancer. Despite their fundamental importance in brain development and health, we know surprisingly little at the molecular level regarding astrocyte specification, growth, and functional interactions with neurons or synapses. We have recently made the exciting discovery that the Drosophila embryonic, larval, and adult nervous system houses a novel cell type that appears strikingly similar to mammalian astrocytes by morphological, functional, and molecular criteria. For example, fly astrocytes are only found in synapse-rich regions of the brain where they acquire a highly branched morphology, they associate closely with synapses, tile with one another to occupy unique spatial domains in the CNS, and express neurotransmitter transporters and metabolizing enzymes (e.g. EAATs, glutamine synthetase, and GABA transporters). This proposal aims to use the powerful array of molecular-genetic tools available in Drosophila, along with a number of astrocyte-specific tools we have generated, to explore fundamental questions in astrocyte biology. In this project we will (Aim1) characterize astrocyte morphology, synaptic association, polarity, and the cell-cell interactions that sculpt astrocyte architecture; (Aim 2) determine the role of the Heartless FGF receptor signaling pathway in promoting astrocyte morphogenesis and synaptic association, and (Aim 3) perform the first forward genetic screen for mutants affecting astrocyte development. We expect our work will provide exciting new insights into the molecular and cellular mechanisms regulating astrocyte development and growth control in vivo, and be highly informative in forwarding our understanding astrocyte development and dysfunction in humans. PUBLIC HEALTH RELEVANCE: Astrocytes are the most abundant cell type in the human brain and have emerged as key regulators of brain development, function, and maintenance. Astrocyte dysfunction results in devastating neurological conditions including periventricular leukomalacia, amyotrophic lateral sclerosis, and gliomas, one of the most deadly forms of cancer. Our work will provide fundamental knowledge about how astrocytes develop and regulate their growth in the brain, and is expected to provide critical insights into how defects in astrocyte growth or function cause disease.

Nervous system injury can have devastating long-term effects on brain or nerve function, yet signaling pathways that regulate nervous system responses to injury, especially in early acute phases, remain poorly defined. In our previous work we sought to identify molecules required to drive axon degeneration after axotomy and identified dSarm/Sarm1 as a key signaling molecule that drives axon auto-destruction. In dsarm/Sarm1 null mutant flies or mice, severed distal axons do not degenerate and remain morphologically intact for weeks after injury. Understanding how dSarm/Sarm1 signals in axons is now a major focus for the field, but the vast majority of studies have focused on the final outcome of axotomy?axonal degeneration?which occurs many hours to days after axotomy. In preliminary work we discovered that nerve injury leads to rapid changes (within 2-3 hrs after injury) in axon transport in both severed axons and adjacent intact neurons, and a suppression of sensory signal transduction in intact neurons throughout the nerve. We wish to understand how injury signals spread throughout the nerve so quickly to activate these response (which we refer to as Phase 1 responses), and the roles that neurons and glia play in this process. Interestingly, we found that components of the dSarm signaling pathway, the Ca2+-driven Unc-76?Cacophony?CamK-II?dSarm signaling pathway, and components of the MAPK pathway play important roles within 3 hrs after injury to alter axonal cell biology and function. In addition, we found that the glial receptor Draper/MEGF10, functions in glia to activate Phase 1 responses in intact neurons (but not severed neurons) within 3 hrs after injury. In Aim 1 we will characterize this novel role for dSarm/Sarm1 and the axon death signaling machinery in regulation of early (Phase 1) responses in intact neurons and severed axons in a simple, genetically-tractable injured nerved, and how these signaling events alter neurophysiology. In Aim 2 we will perform similar studies to explore a novel role for the Unc-76?Cacophony?CamK-II?dSarm signaling pathway and MAPK signaling in axonal Phase 1 responses to nerve injury. In Aim 3 we will determine how nerve injury severity regulates neuronal and glial responses to injury, and how the Draper signaling pathway helps spread injury signals along a nerve to modulate nerve-wide changes in axon physiology. This work will provide important new insights into how axon death signaling molecules regulate acute responses to nerve injury, identify new molecules involved in injury signaling (Unc-76, Cacophony, CamK-II), clarify how MAPK signaling drives changes in axon biology after injury, and delineate exciting new roles for Draper/MEGF10 during the acute window of nerve responses to injury. Given that dSarm/Sarm1 and Draper/MEGF10 signaling pathways (and their functional roles) are highly conserved, our work will illuminate fundamental mechanisms of nervous system injury signaling that should have high relevance to human neural injury and neurological disease.

Summary The nervous system is the most complex tissue in the human body. The formation and maintenance of this amazing structure entails sophisticated mechanisms that drive the specification of appropriate cell fates in along the spatial and temporal axes, and the formation and fine-tuning of highly specific cell-cell contacts that are crucial for organisms to properly sense and respond to their environment. Alterations in normal nervous system development can lead to devastating neurological disorders including Autism, Schizophrenia, and neurodegenerative disease. With the advent of new molecular approaches including whole genome sequencing, CRISPR/Cas9, and imaging techniques including CLARITY, multiphoton microscopy, and ultra- resolution microscopy, the field of developmental neurobiology is making tremendous leaps forward. The 2016 Neural Development Gordon Research Conference (GRC) in Newport, Rhode Island, will bring together an international group of scientists that have made breakthroughs in our understanding of nervous system development and significantly advanced the field. This meeting has been held biennially since 1981 and has remained the premier meeting in the field for the presentation of exciting new advances in the field, and for trainees, young investigators, and senior colleagues, to interact extensively in formal and informal settings. As with many GRCs the meeting is centered around talks and poster sessions, with extensive time for social and scientific interactions in the afternoons. Topics covered will include neural stem cell biology, specification and morphogenesis of neurons and glia, synaptogenesis, neural circuit refinement, and the mechanisms of neural dysfunction in disease. Our initial slate of speakers is an excellent mix of very promising young (10) and mid career scientist (11), along with outstanding senior researchers (13). The meeting is also designed to additionally highlight recent technical advances that have rapidly propelled the field forward, and a diversity of experimental approaches and model systems (e.g. C. elegans, Drosophila, zebrafish, mammals) will be represented. Several short talks by participants will be selected from submitted poster abstracts, and these will be targeted toward trainees and young investigators. Additional features unique to this meeting are lunchtime discussion with trainees on career relevant issues, and our first Power Hour (sponsored by the GRC to promote the mentoring of women in science). The main conference will be preceded by a Gordon Research Seminar, a 2 day event that is organize and run by trainees, where except for the keynote address all talks and posters are given by trainees?our first GRS two years ago added tremendous value the the GRC for the trainees. We anticipate this meeting will foster extensive interactions and collaborations between scientists at all stages of their careers, expose all attendees to exciting new breakthroughs in the field, and let us frame the next exciting set of questions to advance our understanding of nervous system assembly.

? DESCRIPTION (provided by applicant): Specific Aims The major goal of this proposal is to understand mechanisms by which astrocytes interact with neurons to modulate neural circuit activity and animal behavior. Many studies over the past two decades have suggested that astrocytes respond directly to neurotransmission and in turn signal back to neurons to regulate circuit activity. Definitive in vivo examples have remained elusive, but if astrocytes indeed receive synaptic information and feedback to modulate neuronal activity it is imperative that we identify and define these mechanisms-astrocyte control of neuronal activity could represent a previously unappreciated but prominent mechanism for regulating information processing in the brain and animal behavior. We recently made the exciting observation that in live preparations Drosophila astrocytes exhibit rhythmic fluctuations in intracellular Ca2+ similar to those observed in astrocytes of awake behaving mice. Astrocyte Ca2+ signaling in larvae is not altered by application of glutamate, GABA, or acetylcholine, but is potently stimulated by octopamine (OA), the invertebrate equivalent of norepinephrine (NE). Intriguingly, NE was recently found to stimulate astrocytic Ca2+ signaling in mice, although the NE site of action (e.g. neurons, astrocytes, or both) remains unclear, molecules required in astrocytes for NE-induced Ca2+ signals have not been identified, and their physiological roles remain enigmatic. Furthermore, using forward genetic approaches we identified the G protein-coupled octopamine-tyramine receptor (Oct-tyrR) and the TRP channel Waterwitch (Wtrw) as novel, astrocyte-expressed molecules required for activation of OA-induced astrocyte Ca2+ transients. Remarkably, depletion of Wtrw from astrocytes leads to defects in both olfaction and touch- induced startle responses, implicating Wtrw and astrocyte Ca2+ signaling in animal behavior. Our identification of Oct-tyrR and Wtrw as novel critical regulators of astrocyte Ca2+ signaling opens the door to an exploration of the functional significance astrocyte Ca2+ signaling in vivo, which is proposed herein. Our work will define new components of the astrocyte Ca2+ signaling machinery and their importance in astrocyte-neuron signaling events (Aim 1); determine how astrocyte Ca2+ signaling events are regulated by neural circuits and how they reciprocally modulate neuronal activity in vivo (Aim 2); and explore the role of astrocyte Ca2+ signaling molecules in simple sensory-driven behaviors, circadian and arousal behaviors, and a touch induced startle response.

Abstract Mitochondria are integral to neuronal health. Subsequently, deficits in mitochondrial function contribute to a wealth of neurodegenerative diseases, where axonal dysfunction and die back usually precedes cell body demise. However, we know relatively little about the basic biology of mitochondrial biogenesis, morphological changes, transport, or function in axons in vivo. The discovery and characterization of new molecules regulating fundamental aspects of mitochondrial biology in axons may `open the door' to entirely new lines of research in neurodegenerative disease. In this proposal we aim to discover new regulators of mitochondria function in the axon using a novel and high throughput unbiased forward genetic screening approach recently developed in the lab. This approach allows us to assay mitochondrial morphology, number, and distribution in axons with unprecedented single axon and single mitochondrion resolution in vivo. Newly identified mitochondrial genes will then be characterized using an array of new tools we have optimized for mitochondrial studies in Drosophila, and we will determine precisely how mitochondrial physiology has been altered in vivo. We will also genetically determine how novel mitochondrial regulating genes function in defined pathways to control mitochondrial maintenance. Given that mitochondrial health and function is tightly correlated with neurodegenerative disease, it is likely that a number of these genes will play causal and/or accessory roles in neurodegeneration. We will therefore also investigate whether these novel mitochondria associated molecules have an exacerbated phenotype in dopamine neurons, since they selectively degenerate in Parkinson's disease (PD), a condition where mitochondrial dysfunction and oxidative stress is thought to play a fundamental role in disease progression. Functional conservation of these new molecules will then be assayed in mammalian neurons in vitro. This effort represents (to the best of our knowledge) the first high through forward genetic screen for molecules required for mitochondrial transport to and maintenance in axons. Thus a wealth of novel regulators of neuronal mitochondria, which have potential roles in neurological disease, await identification.

Project summary Glial ensheathment of axons is a conserved feature of nervous systems that is essential for proper nervous system function. Impairment or loss of axonal wrapping underlies many debilitating conditions including multiple sclerosis, leukodystrophies, peripheral neuropathies, and CMT diseases. Despite many years of work our understanding of the molecular pathways that control glial development, glial-axon communication, and ensheathment of long axons, including myelination, is far from complete. Our understanding of non-myelinating forms of axon ensheathment is particularly sparse, despite the fact that the majority of peripheral axons (~70%) in humans are unmyelinated and encased by Remak Schwann cells. To address this gap in our understanding we propose to use the genetically tractable model Drosophila to characterize novel molecular mechanisms that promote glial ensheathment of axons and to study the functional roles of non-myelinating ensheathment in axon health and function in vivo. In Drosophila, specialized glia called wrapping glia (WG) ensheath peripheral axons in a manner closely resembling vertebrate Remak SCs. Recent studies (including our own preliminary data) have found that many genes that control the formation of vertebrate myelin also control axon ensheathment by WG in the fly, supporting strong molecular conservation between these forms of ensheathment. We have taken advantage of the fly to conduct a large-scale RNAi screen for novel regulators of ensheathment, and have identified a number of exciting new genes required for glial ensheathment of axons. One candidate to emerge from the screen, discoidin domain receptor (Ddr), encodes an evolutionarily conserved receptor tyrosine kinase activated by collagens. We show that loss of Ddr in WG results in profound defects in axonal ensheathment: although WG can grow longitudinally along the nerve they fail to insert processes between bundled axons to sort and ensheath them. Intriguingly, murine Ddr1 is highly expressed in oligodendrocytes and detailed expression profiling reveals that mDdr1 expression increases at the onset of wrapping during development and with the initiation of remyelination after injury, but functional roles for mDdr in ensheathment or myelination has not been investigated. Our preliminary work has also identified the Type XV/XVIII collagen homolog Multiplexin as required for axon ensheathment, possibly by acting as a ligand for Ddr. In Aim 1 we will characterize the role of Ddr in promoting axonal ensheathment, determine its autonomy of action, and perform a structure function analysis to define key aspects of Ddr signaling in vivo. In Aim 2 we will investigate the role of Mp in driving ensheathment and directly test our model that Mp acts in an autocrine fashion to activate the Ddr receptor on WG. Finally, in Aim 3 we will take advantage of the many genes identified in the screen that have mild to strong ensheathment defects to probe the function of non-myelinating ensheathment on neuronal health and physiology using behavioral assays and in vivo physiological studies. Our work will define the mechanistic basis of Ddr and Mp signaling during nerve assembly and glial ensheathment of axons, and help define the enigmatic but essential functions of non-myelinating forms of ensheathment in complex nervous systems.

SUMMARY Astrocytes are the most abundant glial cell type in the human brain and are critical for central nervous system (CNS) development and function. Mature astrocytes are unusually elaborate cells, with an intricate and ramified morphology. Their numerous fine cellular processes interact closely with synapses, neuronal cell bodies, axons, blood vessels, and other glial cells throughout the CNS. Through these interactions, astrocytes fulfil diverse functions to support and enhance neuronal activity, maintain CNS homeostasis, and modulate circuits. Underscoring the importance of proper astrocyte development, defects in astrocyte growth or loss of astrocyte complexity are implicated in many neurological diseases, including Alexander's disease, autism, and epilepsy. However, it remains poorly understood how astrocytes develop their intricate morphological associations and regulate neural circuit function. Our long-terms goals are to understand how astrocyte acquire their remarkable morphology, target their processes to synapses, and use these cell-cell contacts to modulate brain function. We recently performed a genetic screen in Drosophila to identify new regulators of astrocyte development, and uncovered a novel gene, Trapped in endoderm 1 (Tre1), as required for astrocyte morphogenesis. We find that loss of Tre1 leads to severely reduced astrocyte complexity in vivo, resulting in decreased infiltration of the synaptic neuropil. Tre1 encodes a G protein-coupled receptor (GPCR) with no known function in the CNS. This proposal will use a synergistic combination of molecular-genetic tools available in Drosophila and zebrafish along with new tools we have generated and in vivo imaging to: determine how Tre1 regulates astrocyte morphogenesis, function, and animal behavior in Drosophila (Aim 1); elucidate signaling pathways upstream and downstream of Tre1 activation (Aims 1+2); and define the evolutionary conservation of Tre1 in vertebrates (Aim 3). Our work will provide exciting new insights into the mechanisms regulating astrocyte development and function in vivo and lay the foundation for understanding astrocyte growth and dysfunction in human disease.